Reconstitution of non-ionic monoalkyl amphiphile-cholesterol vesicles by dilution of lipids-octylglucoside mixed micelles

Abstract

Kinetic aspects of the formation of non-ionic surfactant vesicles (NSV) using the mixed micelle dilution procedure are examined. Mixed micelles composed of a mixture of lipids, i.e. diglycerol hexadecylether (C 16G 2), cholesterol (CHOL), dicetylphosphate (DCP) and detergent, octylglucoside (OG), were diluted with detergent-free buffer added either instantaneously or progressively at different rates ranging from 3.47 × 10 −2 to 6.94 × 10 −4 ml/min. The resulting particles were analysed by quasielastic light scattering (QELS), high performance liquid chromatography (HPLC) on gel exclusion column and cryogenic transmission electron microscopy (cryo-TEM). NSV exhibit mean diameters (MD) varying from 100 to 600 nm depending on the kinetics of OG removal. When the dilution of mixed micelles is instantaneous the vesicles are characterized by a spherical shape and MD values close to 100 nm. They show narrow size distribution and stability for 2 months. NSV recovered with progressive micelle dilution, at fast buffer addition rates (3.47 × 10 −2 and 1.39 × 10 −2 ml/min) exhibit MD values of 170–240 nm, elongated shapes, low polydispersities and 2-month stabilities. When the rate of buffer addition is lowered to 6.94 × 10 −4 ml/min, unstable particles with larger MD values and broad size distributions are obtained. Turbidity monitoring at 350 nm and 25°C was used to characterize the lipids-OG mixed aggregate rearrangements either as a function of time when detergent-free buffer was continuously added to the mixed micelles or after equilibrium setting when the micelles were instantaneously diluted. In the latter case the intermediate aggregates were also analysed by QELS. For continuous dilutions, the molecular composition of aggregates, [OG/lip] agg, as well as the OG concentration in the aqueous medium, [OG] bulk, were determined at the break points observed on the plots of optical density (OD) versus total OG concentration ([OG] tot). [OG/lip] agg and [OG] bulk values are independent of the rate of buffer addition, suggesting that the micelle to NSV transition is not mainly limited by the kinetics of the molecular processes involved during detergent removal from the mixed aggregates. The examination of the apparent partition coefficient of OG between the aqueous phase and the lipidic aggregates shows, however, that OG depletion from the bilayered structures is more difficult than its elimination from the mixed micelles. QELS analysis of the intermediate lipids-detergent aggregates, performed with time, demonstrates very slow supramolecular rearrangements during the vesicle closure. These rearrangements explain the significant increase in both size and polydispersity of the final vesicles observed with slow rates of buffer addition.